Studies on Developmental Genetic Programs of Early Embryogenesis in Chicken

Abstract

this maternal gene expression regulated the cleavage period and was replaced by bi-allelic expression after MZT. Our results demonstrate that the feasible mechanism in avian zygotes activating only maternal alleles to inhibit disproportionate genome contribution or genetic instability due to inattentive transcription of paternal alleles.

Finally, we discovered the avian-specific small heat shock proteins in stress-tolerant blastoderm. We found that chicken HSP25 was expressed especially in blastoderm and highly upregulated during low temperature storage. Multiple alignment, phylogenetic trees and the expression in blastoderms of Japanese quail and zebra finch showed homologs of HSP25 were conserved in avian species. After knockdown of chicken HSP25 using siRNAs in blastodermal cells, pluripotency marker genes significantly decreased. Furthermore, this loss of function studies have demonstrated that chicken HSP25 is associated with anti-apoptotic, anti-oxidant and pro-autophagic functions in chicken blastodermal cells. Collectively, avian HSP25 is important in association with the very first line of cellular defense against environmental stresses, and protection of future embryonic cells in avian blastoderm.

Based on the researches, we provide the extensive early development such as ZGA, MZT, signaling pathways in an avian manner from the first transcriptomic approach. Furthermore, unlike the relatively conserved second-wave ZGA, the first-wave ZGA is showing the variable characteristics in each species, which may be due to evolutionary outcomes. Also, the avian-specific HSP25 is the constitutive protector in avian blastoderm dormancy. Our results will filling the gap for comparative studies among the species in developmental and evolutionary respects as a valuable resources for future availability, as well as contribute to comprehensive understanding early avian embryogenesis.

Avian species have been utilized as valuable model system for several studies, especially for embryology. Due to their important position among the vertebrates in the comparative genomics of vertebrate species and wide relevance across many research fields, Bird10K project (B10K) was initiated during 2014~2015. As a part of this project, a phylogenetic hierarchy of avian species and the comparative genomics for flight and functional adaptations were generated. Although historically significant and theoretically excellent, there is no genome-wide transcriptomic data investigating the initial and critical events of avian embryogenesis, mainly because of practical difficulties in accessing the pre-oviposited embryos. In this regard, we presented the first whole transcriptome data of pre-oviposited embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X using a non-invasive method in chicken. Furthermore, our multi-omics approaches investigated the first transcriptional activation upon fertilization in chicken. Also, we identified avian-specific small heat shock protein, HSP25, and found its putative roles in the suspended early development, avian blastoderm dormancy.

The first study was performed to generate whole-transcriptomic information of early chicken embryos and analysis. In this study, a total of 137 pre-oviposited embryos from ovary and oviduct were collected, and RNA sequencing (RNA-seq) was performed in chicken. From these results, two waves of chicken zygotic genome activation (ZGA) governed the distinct developmental programs, such as Notch, MAPK, Wnt, and TGF-beta signaling, between cleavage and area pellucida formation period, separately. Furthermore, EGK stages of chicken were mapped into counterparts in human and mouse, and highlighted the chicken-specific features of signaling pathways, as well as the gradually analogous expression pattern after ZGA. These findings provide a genome-wide understanding of avian embryogenesis and comparative studies among amniotes.

Based on the generated whole-transcriptome sequencing, we identified maternal-to-zygotic transition (MZT), which is a critical process for establishing embryonic identity throughout the vertebrates. Based on the co-expression analysis on the RNA-seq data according to semi-supervised clustering, two waves of ZGA-mediated MZT were observed across the intrauterine stages and transcriptional and translational dynamics were associated. Furthermore, the transitions were found according to the distinct developmental characteristics between cleavage and area pellucida formation period functionally. Finally, epigenetic reprogramming and miR-302s expression suggest that the definite MZT in EGK.VIII during early chicken development. Our study is expected to provide an evolutionary link among vertebrates in perspective of MZT regulation.

Induced pluripotent stem cells (iPSCs) and extended pluripotent stem cells (EPSCs) were generated in mammals, based on studies of the factors with transcriptional regulation in oocytes and early embryos. However, using well-known mammalian transcription factors (TFs) without examining the factors during early development in birds, the iPSC-like cells in avian species have not been fully reprogrammed. In the third study, we characterized the transcriptional transition and related TFs during early chicken development based on a transcriptomic analysis. As a result, two waves of transcriptional activation were found and the TFs were provided in each wave prior to cleavage and area pellucida formation during intrauterine development. Our results contribute to identifying a number of reprogrammable TFs and demonstrate that the cleavage stage, showing a similar state with totipotency compared to mammals and higher-potent features than area pellucida formation period, is the pursuit of reprogramming in birds.

For looking more deeply into ZGA in avian species, we investigated the detailed insight into the onset of genome activation in chicken. Our transcriptomic analysis of early chicken embryos clarified two waves of transcriptional activation after fertilisation and Eyal–Giladi and Kochav Stage V (EGK.V) at both the genome-wide and base resolution of intronic regions. Furthermore, we determined the allelic expression based on breed-specific single nucleotide polymorphisms (SNPs) and traced the first-wave transcripts, accomplished via a multi-omics approach using genome resequencing of parents and whole-transcriptome analysis of their individual embryos. Surprisingly, maternal genome activation (MGA) was exclusively found in the zygote stage, regardless of the presence of haploid genomes in male PN and supernumerary sperm in the egg